U.S. patent application number 09/899807 was filed with the patent office on 2002-08-08 for cancer therapeutics involving the administration of 2-methoxyestradiol and an agent that increases intracellular superoxide anion.
Invention is credited to Feng, Li, Huang, Peng, Plunkett, William K..
Application Number | 20020106348 09/899807 |
Document ID | / |
Family ID | 26912066 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020106348 |
Kind Code |
A1 |
Huang, Peng ; et
al. |
August 8, 2002 |
Cancer therapeutics involving the administration of
2-methoxyestradiol and an agent that increases intracellular
superoxide anion
Abstract
The instant invention discloses methods and compositions for the
treatment of cancer. The invention relates methods and compositions
for specifically targeting free radical accumulation as a means of
preferentially eliminating neoplastic cells. The combination of an
SOD inhibitor (2-methoxyestrdiol) with free radical-producing
agents results in a means of eliminating tumor cells through the
accumulation of intracellular superoxide anion.
Inventors: |
Huang, Peng; (Houston,
TX) ; Plunkett, William K.; (Houston, TX) ;
Feng, Li; (Sugar Land, TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
A REGISTERED LIMITED LIABILITY PARTNERSHIP
600 CONGRESS AVENUE, SUITE 2400
AUSTIN
TX
78701
US
|
Family ID: |
26912066 |
Appl. No.: |
09/899807 |
Filed: |
July 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60217589 |
Jul 12, 2000 |
|
|
|
Current U.S.
Class: |
424/85.1 ;
514/15.1; 514/182; 514/19.3; 514/20.9; 514/34; 514/72 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61K 45/06 20130101; A61P 35/00 20180101; A61K 31/565 20130101;
A61K 31/565 20130101 |
Class at
Publication: |
424/85.1 ; 514/8;
514/34; 514/72; 514/182 |
International
Class: |
A61K 038/19; A61K
038/08; A61K 031/704; A61K 031/56 |
Claims
What is claimed is:
1. A method of killing a cell comprising: a) contacting said cell
with a first composition comprising an agent that increases
intracellular O.sub.2.sup.-; and b) contacting said cell with a
second composition comprising 2-methoxyestradiol.
2. The method of claim 1, wherein said cell is a cancer cell.
3. The method of claim 2, wherein said cancer cell is derived from
a solid tumor.
4. The method of claim 2, wherein said cancer cell is a leukemia
cell.
5. The method of claim 1, wherein said cell is a human cell.
6. The method of claim 1, wherein said compound that increases
intracellular O.sub.2.sup.- is rotenone.
7. The method of claim 1, wherein said compound that increases
intracellular O.sub.2.sup.- comprises bleomycin.
8. The method of claim 1, wherein said compound that increases
intracellular O.sub.2.sup.- comprises daunorubicin.
9. The method of claim 1, wherein said compound that increases
intracellular O.sub.2.sup.- comprises epirubcin.
10. The method of claim 1, wherein said agent that increases
intracellular O.sub.2.sup.- comprises TNF-alpha.
11. The method of claim 1, wherein said agent that increases
intracellular O.sub.2.sup.- comprises heat.
12. The method of claim 1, wherein said agent that that increases
intracellular O.sub.2.sup.- comprises an arsenate.
13. The method of claim 1, wherein said agent that that increases
intracellular O.sub.2.sup.- comprises a retinoic acid
derivative.
14. The method of claim 1, wherein the administration of said first
composition and said second composition is substantially
concurrent.
15. The method of claim 1, wherein the administration of said first
composition is subsequent to the administration of said second
composition.
16. The method of claim 1, wherein the administration of said first
composition is prior to the administration of said second
composition.
17. The method of claim 1, wherein said first and said second
compositions are combined in a single formulation.
18. A method of treating cancer comprising administering to a host
a composition comprising 2-methoxyestradiol and an agent that
increases intracellular O.sub.2.sup.-.
19. The method of claim 18, wherein said agent that increases
intracellular O.sub.2.sup.- is rotenone.
20. The method of claim 18, wherein said agent that increases
intracellular O.sub.2.sup.- comprises bleomycin.
21. The method of claim 18, wherein said agent that increases
intracellular O.sub.2.sup.- comprises daunorubicin.
22. The method of claim 18, wherein said agent that increases
intracellular O.sub.2.sup.- comprises epirubcin.
23. The method of claim 18, wherein said agent that increases
intracellular O.sub.2.sup.- comprises TNF-alpha.
24. The method of claim 18, wherein said agent that increases
intracellular O.sub.2.sup.- comprises heat (hyperthermia).
25. The method of claim 18, wherein said agent that that increases
intracellular O.sub.2.sup.- comprises an arsenate.
26. The method of claim 18, wherein said agent that that increases
intracellular O.sub.2.sup.- comprises a retinoic acid
derivative.
27. The method of claim 18, wherein said host is a human.
28. The method of claim 18, wherein the administration of said
first composition and said second composition is substantially
concurrent.
29. The method of claim 18, wherein the administration of said
first composition is subsequent to the administration of said
second composition.
30. The method of claim 18, wherein the administration of said
first composition is prior to the administration of said second
composition.
31. The method of claim 18, wherein said first and said second
compositions are contained within a pharmaceutically acceptable
composition.
32. The method of claim 31, wherein said pharmaceutically
acceptable composition includes a pharmaceutically acceptable
carrier.
33. The method of claim 31, wherein said pharmaceutical composition
is formulated for oral administration.
34. The method of claim 31, wherein said pharmaceutical composition
is formulated for parenteral administration.
35. The method of claim 31, wherein said pharmaceutical composition
is formulated for administration by injection.
36. The method of claim 18, wherein said host has cancer.
37. The method of claim 36, wherein said cancer is a solid
tumor.
38. The method of claim 36, wherein said cancer is a leukemia.
39. The method of claim 18, wherein said first and said second
compositions are combined in a single formulation.
40. A composition comprising 2-methoxyestradiol and a second
compound that increase intracellular O.sub.2.sup.-.
41. The composition of claim 40, wherein said compound that
increases intracellular O.sub.2.sup.- comprises rotenone.
42. The composition of claim 40, wherein said compound that
increases intracellular O.sub.2.sup.- comprises bleomycin.
43. The composition of claim 40, wherein said compound that
increases intracellular O.sub.2.sup.- comprises daunorubicin.
44. The composition of claim 40, wherein said compound that
increases intracellular O.sub.2.sup.- comprises epirubicin.
45. The composition of claim 40, wherein said agent that that
increases intracellular O.sub.2.sup.- comprises an arsenate.
46. The composition of claim 40, wherein said agent that that
increases intracellular O.sub.2.sup.- comprises a retinoic acid
derivative.
47. The composition of claim 40, wherein said composition is a
pharmaceutically acceptable composition.
48. The composition of claim 40, wherein said compound that
increases intracellular O.sub.2.sup.- comprises tumor necrosis
factor-alpha.
49. The composition of claim 48, wherein said pharmaceutically
acceptable composition includes a pharmaceutically acceptable
carrier.
50. The composition of claim 48, wherein said pharmaceutical
composition is formulated for oral administration.
51. The composition of claim 48, wherein said pharmaceutical
composition is formulated for parenteral administration.
52. The composition of claim 48, wherein said pharmaceutical
composition is formulated for administration by injection.
Description
BACKGROUND OF THE INVENTION
[0001] This application claims priority to provisional application
No. 60/217,589 filed Jul. 12, 2000, herein incorporated by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
cancer therapeutics. More particularly, it concerns compositions
and methods for increasing intracellular O.sub.2.sup.- while
inhibiting the activity of superoxide dismutase.
DESCRIPTION OF RELATED ART
[0003] Superoxide anion, O.sub.2.sup.-, is a toxic reactive oxygen
intermediate produced by the transfer of a single electron to
O.sub.2. Superoxide anion is formed in the process of cellular
respiration, specifically during oxidative phosphorylation. While
oxidative pathways have evolved to minimize O.sub.2.sup.-
production, a small amount of superoxide anion is unavoidably
formed during the metabolic reduction of oxygen.
[0004] Reactive oxygen intermediates have been implicated in a
number of human degenerative processes, diseases and syndromes,
including the following: mutagenesis, cell transformation and
cancer; atherosclerosis, arteriosclerosis, heart attacks, strokes
and ischaemia/reperfusion injury; chronic inflammatory diseases,
such as rheumatoid arthritis, lupus erythematosus and psoriatic
arthritis; acute inflammatory problems, such as wound healing;
photo-oxidative stresses to the eye, such as cataract;
central-nervous-system disorders, such as certain forms of familial
amyotrophic lateral sclerosis, certain glutathione
peroxidase-linked adolescent seizures, Parkinson's disease and
Alzheimer's dementia; and a wide variety of age-related disorders,
possibly including factors underlying the aging process itself.
Reactive oxygen intermediates are known to produce a variety of
pathological changes through lipid peroxidation and DNA damage. The
common pathway of tissue injury mediated by these toxic oxygen
metabolites involves the destruction of membranes, proteins, and
nucleic acids.
[0005] Antioxidant enzymes protect cells from the toxic effects of
high concentrations of reactive oxygen species generated during
cellular metabolism. Superoxide dismutases (SOD) are metalloenzymes
that catalyze the dismutation of superoxide ion into oxygen and
hydrogen peroxide:
O.sub.2.sup.-+O.sub.2.sup.-+2H.sup.+
.fwdarw.H.sub.2O.sub.2+O.sub.2
[0006] The enzymes scavenge superoxide anion and act as a primary
defense system against oxidative stress in body. Three classes of
SODs have been described, each characterized by the catalytic metal
at the active site, namely, Cu/Zn-SOD, Mn-SOD, and Fe-SOD. Cu/Zn
enzymes are found primarily in eukaryotes, Fe-SOD is found mainly
in prokaryotes and Mn-SOD crosses the entire range from prokaryotes
to eukaryotes. The CuZn-SOD is localized in the cytosol and
nucleus, while Mn-SOD is located within the mitochondrial matrix.
It has been widely recognized that such enzymes provide a defense
system that is essential for the survival of aerobic organisms.
[0007] Carcinogens and tumor promoters are known to decrease the
cellular activity of superoxide dismutase. Consequently, many
neoplastic cells have reduced superoxide dismutase activity. Tumor
cells nevertheless, carry out normal oxidative pathways and thus
accumulate reactive oxygen intermediates at a rate at least equal
to normal cells.
[0008] The critical function of the dismutation of O.sub.2.sup.-
makes SOD an attractive target for pharmacological intervention.
Although several small molecules, including cyanide ion (CN.sup.-),
hydroxyl ion (OH.sup.-), and azide ion (N.sub.3.sup.-), inhibit SOD
by competing with O.sub.2.sup.- at the catalytic site, these
chemicals are highly toxic and their potential for cancer therapy
is limited.
SUMMARY OF THE INVENTION
[0009] The instant invention addresses a noted deficiency in the
art by providing therapeutic compositions and methods for the
treatment of cancer. The invention discloses that
2-methoxyestradiol inhibits SOD and compromises the cell's ability
to eliminate superoxide anion, and that the combination of
2-methoxyestradiol with an agent or agents that increases reactive
oxygen intermediates within a cell results in an enhanced ability
to kill cells, specifically cancer cells. While both classes of
agents may be used independently as cancer therapeutics, the
instant invention discloses that the mechanism-based combination of
compounds produces a synergistic effect that dramatically increases
the tumoricidal and/or anti-neoplastic efficacy of each
compound.
[0010] In one embodiment, the instant invention therefore comprises
a method of killing a cell comprising contacting the cell with a
first composition comprising an agent that increases intracellular
O.sub.2.sup.- and with a second composition comprising
2-methoxyestradiol. Alternate embodiments of this method
contemplate that the two compositions may be provided substantially
concurrently or the 2-methoxyestradiol may be delivered prior to or
subsequent to administration of the agent that increases
intracellular O.sub.2.sup.-. In a further embodiment, the two
compositions may be combined in a single formulation.
[0011] A variety of chemical compounds and physical modalities are
known to increase the intracellular concentration of reactive
oxygen intermediates, i.e., O.sub.2.sup.-. In a particular
embodiment of the invention, the compound administered to increase
intracellular O.sub.2.sup.- will be rotenone, tumor necrosis
factor-alpha, cisplatin, bleomycin, an arsenate (i.e. arsenic
trioxide), a retinoic acid derivative such as all-trans retinoic
acid, or an anthracycline. Where the compound is an anthracycline,
it may be doxorubicin, daunorubicin, epirubcin or daunomycin. A
physical modality administered to increase intracellular
O.sub.2.sup.- will be heat (hyperthermia), ultraviolet rays, X-rays
or .gamma.-rays.
[0012] It is contemplated that the method of the instant invention
may be used in a variety of applications to kill a specific cell.
The instant invention is equally applicable in either an in vivo or
in vitro environment. In another embodiment of the invention, the
cell to be killed is a cancer cell. In alternate aspects of the
invention, the cancer cell is derived from a solid tumor or is a
leukemia cell. In a still further aspect, the cell is a human
cell.
[0013] An alternate embodiment of the invention relates a method of
treating cancer comprising administering to a host a first
composition comprising 2-methoxyestradiol and a second composition
comprising an agent that increases intracellular O.sub.2.sup.-.
[0014] The invention is further contemplated to encompass a
composition comprising 2-methoxyestradiol and an agent capable of
increasing the intracellular concentration of O.sub.2.sup.-. In a
specific embodiment of this aspect of the invention, the compound
administered to increase intracellular O.sub.2.sup.- will be
rotenone, tumor necrosis factor-alpha, cisplatin, bleomycin, an
arsenate (i.e. arsenic trioxide), a retinoic acid derivative such
as all-trans retinoic acid, or an anthracycline. Where the compound
is an anthracycline, it may be doxorubicin, daunorubicin, epirubcin
or daunomycin. A physical agent administered to increase
intracellular O.sub.2.sup.- will be heat (hyperthennia),
ultraviolet rays, X-rays or .gamma.-rays.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0016] FIG. 1: Chemical Structure of 2-methoxyestradiol.
[0017] FIG. 2: Chemical Structure of rotenone.
[0018] FIG. 3: Effect of 2-methoxyestradiol on Survival of K562
Cells (MTT, 72 h).
[0019] FIG. 4: Effect of 2-methoxyestradiol on the Survival of Raji
Cells (MTT, 72 h).
[0020] FIG. 5: Induction of Apoptosis by 2-methoxyestradiol in
Primary Leukemia Cells.
[0021] FIG. 6: Effect of 2-methoxyestradiol (M) and Fludarabine (F)
on the Survival of Primary CLL cells (Patient no. 1, MTT, 72
h).
[0022] FIG. 7: Effect of 2-methoxyestradiol (M) and Fludarabine (F)
on the Survival of Primary CLL cells (Patient no. 2, MTT, 96
h).
[0023] FIG. 8: Effect of 2-methoxyestradiol (M) and Fludarabine (F)
on the Survival of Primary CLL cells (Patient no. 3, MTT, 96
h).
[0024] FIG. 9: Effect of 2-methoxyestradiol on the Growth of Breast
Cancer Cells (MTT, 72 h).
[0025] FIG. 10 Effect of 2-methoxyestradiol on the Growth of Lung
Cancer Cells (MTT, 72 h).
[0026] FIG. 11 Identification of SOD as a Target of
2-methoxyestradiol by Human cDNA Expression Array I.
[0027] FIG. 12: Effect of 2-methoxyestradiol (1 .mu.MM) on SOD
Protein Level in ML-1 and HL-60 Cells.
[0028] FIG. 13: Inhibition of H.sub.2O.sub.2 Production by
2-methoxyestradiol in Whole Cells.
[0029] FIG. 14: Loss of Mitochondria Membrane Potential Induced by
2-methoxyestradiol (2 .mu.M) in HL-60 Cells.
[0030] FIG. 15: Release of Cytochrome c to Cytosol (HL-60,
2-methoxyestradiol, 2 .mu.M).
[0031] FIG. 16: Activation of JNK is not Essential for Apoptosis
Induction by 2-methoxyestradiol (2-methoxyestradiol, 1 .mu.M; FSK,
10 .mu.M, 24 h).
[0032] FIGS. 17A, 17B, 17C, 17D and 17E: Selective cytotoxicity of
2-ME against human leukemia cells.
[0033] FIG. 17A: DNA fragmentation in ML-1 and HL-60 cells
incubated with 1 .mu.M 2-ME.
[0034] FIG. 17B: DNA fragmentation in normal lymphocytes. Lane 1-4,
lymphocytes incubated with 0, 3, 10, and 30 .mu.M 2-ME for 48 h;
lanes 5-8, PHA-stimulated (5 .mu.g/ml) lymphocytes incubated with
0, 3, 10, and 30 .mu.M 2-ME for 48 h.
[0035] FIG. 17C: Effect of 2-ME on normal and PHA-stimulated
lymphocytes (MTT assay, 72 h).
[0036] FIG. 17D: Effect 2-ME on primary leukemia cells and normal
lymphocytes (MTT assay).
[0037] FIG. 17E: Comparison of 2-ME activity in CLL cells (n=31)
and normal lymphocytes (n=9).
[0038] FIGS. 18A, 18B, 18C and 18D: Effect of 2-ME on SOD
expression and free radical metabolism in leukemia cells.
[0039] FIG. 18A: RT-PCR analysis of SOD1 mRNA in 2-ME-treated
cells.
[0040] FIG. 18B: Effect of 2-ME (2 .mu.M) on SOD1 and SOD2 protein
expression in ML-1 and HL-60 cells.
[0041] FIG. 18C: O.sub.2.sup.- accumulation in ML-1 cells treated
with 1 .mu.M 2-ME for 5 h. The dotted lines indicate the mean
fluorescence intensity.
[0042] FIG. 18D: Accumulation of O.sub.2.sup.- in primary CLL cells
treated with 10 .mu.M 2-ME for 24 h.
[0043] FIGS. 19A, 19B, 19C and 19D: Inhibition of SOD by 2-ME.
[0044] FIG. 19A: In vitro assay of SOD1 activity.
[0045] FIG. 19B: Effect of 2-ME on human and bovine CuZnSOD and E.
coli MnSOD.
[0046] FIG. 19C Effect of 2-ME on xanthine oxidase.
[0047] FIG. 19D: Effect of 2-ME on human DNA polymerase .alpha. and
bovine alkaline phosphatase (measured by removal of 5'-phosphate
from [.sup.14C]GMP).
[0048] FIG. 20: Structure of estrogen derivatives, inhibition of
CuZnSOD, and induction of apoptosis in HL-60 cells. The degree of
SOD inhibition: (-), less then 25% inhibition; (+), 25-50%; (++),
50-75%; (+++), 75-100%. Lane (-), control; lanes 1-5, cells treated
with the respectively numbered compounds.
[0049] FIGS. 21A, 21B, 21C 21D, 21E, and 21F:
[0050] FIG. 21A: Effect of SOD1 overexpression on 2-ME-induced
apoptosis. Lane 1, control A2008 cells; lanes 2-3, transduction
with Ad.CuZnSOD for 24 and 48 h; lane 4, control vector (48 h). 10
.mu.M 2-ME was added 24 h after transduction and incubated for
another 48 h before DNA fragmentation assay.
[0051] FIGS. 21B-C: Effect of ectopic expression of SOD1 or SOD2 on
the survival of A2008 cells (MTT assay) and H1299 cells (colony
formation).
[0052] FIG. 21D: SOD antisense S-oligos enhanced the activity of
2-ME. A2008 cells were incubated with S-oligos against SOD1 (SEQ ID
NO: 1, 5'-ACGCACACGGCCTTCGTCGCCATAACT) and SOD2 (SEQ ID NO: 2,
5'-GCACACTGCCCGGCTCAACATGCTG) or the respective scrambled S-oligos
(Scr). SOD protein levels were assayed at 24 h. Lane 1, control;
lane 2, 10 .mu.M each of the scrambled S-oligos; lane 3, 10 .mu.M
each of anti-SOD1 and anti-SOD2 S-oligos. 2-ME was added at 24 h
and incubated for another 48-72 h followed by MTT assay. E) Effect
of anti-SOD1 or random (Rd) S-oligo (10 .mu.M) on the survival of
2-ME-treated H1299 cells (MTT, 72 h). F) Effect of antioxidants on
2-ME-induced apoptosis. Lanes 1 and 5, control HL-60 cells; lanes
2-4, cells treated with 1 .mu.M 2-ME plus 0, 0.03, and 0.1 mM
ambroxol; lanes 6-8, cells treated with 1 .mu.M 2-ME plus 0, 3, and
5 mM N-acetylcysteine.
[0053] FIGS. 22A and 22B:
[0054] FIG. 22A: Accumulation of 2-ME in leukemia cells and normal
lymphocytes (see Methods).
[0055] FIG. 22B: HPLC analysis of extracts from cells incubated
with [3H]2-ME (0.5 .mu.Ci/ml, 5 h). The top two panels are
chromatograms of [.sup.3H]2-ME standard monitored by UV and liquid
scintillation counting, respectively. The lower three panels show
the profiles of radioactivity in extracts of ML-1, HL-60, and CLL
cells, respectively.
[0056] FIG. 23: Biochemical basis of combination strategies: effect
of rotenone on redox status and the activity of
2-methoxyestradiol.
[0057] FIG. 24: Effect of 2-methoxyestradiol and rotenone on
intracellular superoxide contents in HL-60 cells.
[0058] FIG. 25A and FIG. 25B: Synergistic activity of rotenone and
2-methoxyestradiol in HL-60 cells.
[0059] FIG. 26: The synergistic activity of rotenone and
2-methoxyestradiol is cell cycle-independent.
[0060] FIG. 27: Activation of apoptotic cascade by
2-methoxyestradiol and rotenone.
[0061] FIG. 28: Effect of rotenone and 2-methoxyestradiol on
cellular ribonucleotide pools.
[0062] FIG. 29: Caspase-3 activation by oxidized and reduced forms
of cytochrome c in a cell free system.
[0063] FIG. 30: Induction of DNA fragmentation by oxidized and
reduced forms of cytochrome c in isolated nuclei.
[0064] FIG. 31: Effect of 2-methoxyestradiol and rotenone on colony
formation in human lung cancer cells (H1299 cells).
[0065] FIG. 32A, FIG. 32B and FIG. 32C: Suppression of the
cytotoxic effect of 2-methoxyestradiol by an antioxidant
N-acetylcysteine in primary human chronic lymphocytic leukemia
(CLL) cells.
[0066] FIG. 33: Biochemical basis for combination of ionizing
radiation (IR) and 2-methoxyestradiol (2-ME).
[0067] FIG. 34A and FIG. 34B: Synergistic activity of
2-methoxyestradiol (2-ME) and ionizing radiation in human lung
cancer H1299 cells.
[0068] FIG. 35: Effect of 2-methoxyestradiol on the survival of
Lymphocytes from 5 healthy Donors (MTT, 72 h).
[0069] FIG. 36: Effect of 2-methoxyestradiol on the survival of
Lymphocytes from 3 healthy Donors (MTT, 96 h).
[0070] FIG. 37: Effect of 2-methoxyestradiol on the survival of
Leukemia Cells (MTT, 72 h).
[0071] FIG. 38: Inhibition of SOD by 2-methoxyestradiol in
vitro.
[0072] FIG. 39: Effect of 2-methoxyestradiol and arsenate
combination on cell survival in 2-ME-sensitive primary leukemia
cells (CLL-376550) in vitro (MTT assay, 72 h incubation).
[0073] FIG. 40: Effect of 2-methoxyestradiol and arsenate
combination on cell survival in 2-ME-sensitive primary leukemia
cells (CLL-257266) in vitro (MTT assay, 72 h drug incubation).
[0074] FIG. 41: Effect of 2-methoxyestradiol and arsenate
combination on cell survival in 2-ME-resistant primary leukemia
cells (CLL-266050) in vitro (MTT assay, 72 h drug incubation).
[0075] FIG. 42: Effect of 2-methoxyestradiol and arsenate
combination on cell survival in 2-ME-resistant primary leukemia
cells (CLL-384854) in vitro (MTT assay, 72 h drug incubation).
[0076] FIG. 43: Combination of 2-methoxyestradiol of all-trans
retinoic acid caused substantial increase of cellular superoxide
content (flow cytometry analysis), and resulted in a synergistic
activity against leukemia cells from a CLL patient (MTT assay).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0077] The instant invention, provides a therapeutic approach
wherein a compound or compounds that inhibits SOD activity is
administered in combination with an agent or agents that increases
intracellular reactive oxygen intermediate accumulation. The active
O.sub.2.sup.- production and low SOD activity in cancer cells
renders malignant cells highly dependent on SOD for survival and
sensitive to inhibition of SOD. Inhibition of SOD causes an
accumulation of cellular O.sub.2.sup.- and leads to free
radical-mediated damage to mitochondrial membranes, the release of
cytochrome c from mitochondria, and apoptosis of the cancer cells.
The inventors have determined that combining the SOD inhibiting
activity of 2-methoxyestradiol with an agent that increases
intracellular O.sub.2.sup.- results in a markedly enhanced killing
of tumor cells. The instant invention provides compositions and
methods for enhancing the antineoplastic/tumoricidal properties of
an SOD inhibitor by concomitantly increasing intracellular oxygen
radical concentrations with the administration of an additional
agent that increases reactive oxygen intermediates within the
targeted cancer cell.
[0078] A number of inhibitors of SOD activity have been identified.
Unfortunately, the majority are either highly toxic or otherwise
unsuitable for administration in a therapeutic regimen.
2-methoxyestradiol, a human steroid metabolite, is an inhibitor of
superoxide dismutase, including cytosolic SOD1 (CuZn-SOD) and
mitochondrial SOD2 (Mn-SOD), that is suitable for pharmaceutical
use. Treatment of cancer cells with 2-methoxyestradiol causes an
oxidative stress in the cells and triggers apoptosis in the cancer
cells, with the most prominent effect to date observed in human
leukemia cells. The effect is apparently specific to neoplastic
cells, as no apparent cytotoxic effect is observed in normal human
lymphocytes from healthy donors incubated with 2-methoxyestradiol,
suggesting that certain neoplastic cells may depend more upon SOD
for survival than normal cells. A potential added benefit of the
administration of 2-methoxyestradiol, particularly in the context
of leukemia, is that because SOD is an inhibitor of erythroid
progenitor cell cycling, 2-methoxyestradiol may potentially
stimulate the production of red blood cells and thus alleviate the
anemic conditions often associated with leukemia.
[0079] Chemical compounds and physical modalities that enhance the
generation of free radicals by-products, especially oxygen
radicals, are well known in the art. While mechanisms of activity
differ among the various compounds, they all facilitate the
intracellular accumulation of reactive oxygen intermediates that
are ultimately cytotoxic. Beyond a certain threshold concentration,
reactive oxygen species elicit the onset of apoptosis by the
induction of mitochondrial membrane permeability transition and
release of cytochrome c (Lee, et al., 2000).
[0080] The instant invention demonstrates an enhanced killing of
cells previously known to be susceptible to 2-methoxyestradiol
treatment by increasing intracellular O.sub.2.sup.- in the target
cells. The instant invention further contemplates that these
methods and compositions will be effective against cell types
previously deemed to be resistant to 2-methoxyestradiol therapy.
The combination of the SOD inhibitor, 2-methoxyestradiol, with an
agent that effectively increases intracellular O.sub.2.sup.- is
contemplated to be effective in overcoming the ability of cells to
specifically resist either composition delivered independently.
[0081] A. 2-methoxyestradiol
[0082] 2-Methoxyestradiol (1,3,5(10)-estratrien-2,3,17b-triol 2
methylether (C.sub.19H.sub.7O.sub.3)) is a multicyclic estradiol
derivative with a molecular weight of 302.42. The compound has been
previously shown to inhibit the formation of new blood vessels
required by tumors and also to directly inhibit the growth of tumor
cells. 2-methoxyestradiol is commercially available (Research Plus,
Bayonne, N.J.) or may be formulated as described below.
[0083] Methods for the synthesis of estradiol and estradiol
derivatives are well known in the art; see, for example Eder,
(1979), and Oppolzer and Roberts, (1980). Further, methods for
constructing seven membered rings in multi-cyclic compounds are
well established (Nakamuru, et al., 1962, Sunagawa, et al., 1961,
Van Tamelen, et al., 1961, Evans, et al., 1981). The chemical
synthesis of estradiol may be readily modified to include
7-membered rings by making appropriate changes to the starting
materials, so that the process of ring closure results in
seven-membered rings. Known chemical methods facilitate the
modification of estradiol or estradiol derivatives to include the
appropriate chemical side groups according to the invention (The
Merck Index, 1989), pp. 583-584).
[0084] B. Inducers of Reactive Oxygen Intermediates
[0085] 1. Rotenone
[0086] Rotenone,
2R-2.alpha.,6.alpha.,12a.alpha.)-1-2,12,12a-tetra-hydro-8-
,9-dimethoxy-2-(1-meth-ylethenyl)-[1] benzopyrano-[3,4-b]
furo[2,3-h] [1]benzyopyran-6(6aH)-one (C.sub.23H.sub.22O.sub.6)) is
a multicyclic, naturally occurring substance found in the roots and
stems of several tropical plants. Extracts of these plants have
been used for centuries as potent pesticides and insecticides.
Rotenone works by inhibiting mitochondrial electron transport,
resulting in an inability of the cell to use oxygen in the release
of energy during normal body processes. In effect, the cells
suffocate due to lack of oxidative phosphorylation. The cytotoxic
activity of rotenone is attributed to its inhibition of
NADH:ubiquinone oxidoreductase activity, while its potential cancer
chemopreventive effect of has been associated with inhibition of
phorbol ester-induced ornithine decarboxylase (ODC) activity.
Further study suggests that the molecular features of rotenone
essential for inhibiting NADH:ubiquinone oxidoreductase are similar
to those for blocking ODC induction, with the IC.sub.50 values in
the range of 0.8-4 nM. It is proposed that inhibition of
NADH:ubiquinone oxidoreductase activity lowers the level of induced
ODC activity, leading to the antiproliferative effect and
anticancer action (Fang et at, 1998). In the context of the instant
invention, it is contemplated that inhibition of the mitochondrial
electron transport by rotenone will cause an increase of
intracellular superoxide radicals and, when combined with
2-methoxyestradiol to inhibit SOD, will result in a severe
superoxide stress and produce synergistic anticancer activity.
[0087] 2. Tumor Necrosis factor-alpha
[0088] Tumor necrosis factor-alpha (TNF-.alpha.) is a naturally
occurring cytokine involved in a variety of biological processes
including inflammation, cancer cachexia, and regulation of cell
growth and apoptosis. This molecule is known to induce production
of reactive oxygen species and to cause apoptosis in cells. It is
demonstrated that the TNF-alpha-induced apoptosis through
production of superoxide anion, which function as the crucial
mediator for the TNF-alpha-initiated apoptotic pathway
(Moreno-Manzano et al, 2000). TNF-alpha has been used in clinical
trials in a variety of human cancer patients. For example,
combination of TNF-alpha and interleukin-2 significantly improved
the response rate of patients with metastatic renal cell carcinoma
compared to treatment with either agent alone (Bukowski, 2000).
TNF-alpha has also been used in combination with melphalan and
hyperthermia in treating patients with malignant melanoma or breast
cancer, with induction of complete remissions (Robins, 1999).
TNF-alpha is administered intravenously, with a dose range of
50-100 micrograms/m.sup.2. The dose-limiting toxicity include
myelosuppression and thrombocytopenia. It is suggested that high
doses of TNF-alpha may be administered by isolated limb perfusion
to treat unresectable sarcoma and melanoma (Fraser et al., 1999).
In the context of the instant invention, TNF-alpha could be
combined with 2-methoxyestradiol to cause enhanced accumulation of
intracellular superoxide radicals and thus produce synergistic
anticancer activity.
[0089] 3. Bleomycin Bleomycin A.sub.2, Ni
[3-(dimethylsulfonio)propyl]bleo- mycinamide
(C.sub.55H.sub.84N.sub.17O.sub.21S.sub.3), is a glycopeptide member
of the bleomycin peptide family isolated from Streptomyces
verticillus. Bleomycins are known to cause strand scission of DNA
as well as possessing oxygen transferase activity. Strand scission
occurs because of free radical generation from the interaction of
bleomycin, iron, and oxygen. The compounds are employed as
antimicrobial and antineoplastic agents.
[0090] Bleomycin is poorly absorbed across the GI tract and must be
administered parenterally. Bleomycin may be administered
intramuscularly, intravenously, subcutaneously or intrapleurally.
The general dosage employed in treatment regimens is 0.25 to 0.5
units/kg (10-20 units/m.sup.2) intramuscularly, intravenously or
subcutaneously, delivered at weekly or bi-weekly intervals.
Pulmonary toxicity of the drug is dose dependent with a striking
increase in toxicity above 400 units.
[0091] 4. Cisplatin
[0092] Cisplatin, cis-Diamminedichloroplatinum
(Cl.sub.2H.sub.6N.sub.2Pt), is a heavy metal complex containing a
central atom of platinum surrounded by two chloride atoms and two
ammonia molecules in the cis position. Cisplatin cytotoxicity is
based upon a number of alternate effects including DNA binding,
mitochondrial damage, decreased ATPase activity, and altered
cellular transport mechanisms. After entering cells by diffusion,
cisplatin becomes chemically active due to the loss of its chloride
ions by hydrolysis. Free radical intermediates are produced as the
consequence of the chemical reactions. The drug's efficacy is based
upon its stereochemistry, as the trans isomer is not cytotoxic.
Cisplatin is a non-cell cycle specific, bifunctional, alkylating
agent with activity against both solid tumors and lymphoma.
Cisplatin is currently the treatment of choice in many testicular,
bladder, gastric, head and neck, non-small cell lung, ovarian, and
small cell lung cancers.
[0093] Cisplatin is administered primarily through IV infusion
although intra-arterial delivery is also possible. Dosage of
cisplatin varies from 20 mg/m.sup.2 to over 100 mg/m.sup.2
depending upon the nature of the neoplasia treated. Renal and
hematologic function must be assessed prior to the administration
of cisplatin. Dosage in excess of 100 mg/m.sup.2 must be closely
monitored due to the risk of overdosage and platinum toxicity. To
decrease the incidence and severity of nephrotoxicity, hydration
consisting of NS 250 ml/hr for 2-4 hours prior to and post
cisplatin, and maintenance of urine output of 100-150 ml/hr for 24
hours following cisplatin are recommended. In addition,
premedication with antiemetics including a serotonin antagonist and
corticosteroid prevents the severe nausea and vomiting associated
with the drug.
[0094] 5. Anthracycline Derivatives
[0095] The anthracycline antibiotics were initially derived from
the fungus Streptomyces peuceitius var. caesius. Synthetic members
of this family have also been produced. Anthracyclines achieve
their cytotoxic effect by several mechanisms, including: inhibition
of topoisomerase II; intercalation between DNA strands, thereby
interfering with DNA and RNA synthesis; production of free radicals
that react with and damage intracellular proteins and nucleic
acids; chelation of divalent cations; and reaction with cell
membranes. The wide range of potential sites of action may account
for the broad efficacy as well as the toxicity of the
anthracyclines (Young et al., 1985).
[0096] The anthracycline antibiotics have tetracycline ring
structures with an unusual sugar, daunosamine, attached by
glycosidic linkage. Cytotoxic agents of this class all have quinone
and hydroquinone moieties on adjacent rings that permit them to
function as electron-accepting and donating agents.
[0097] a. Doxorubicin
[0098] Doxorubicin hydrochloride, 5,12-Naphthacenedione,
(8s-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexopyranosyl)oxy]-7,-
8,9,10-tetrahydro-6,8,1-trihydroxy-8-(hydroxyacetyl)-1-methoxy-hydrochlori-
de (hydroxydaunorubicin hydrochloride, Adriamycin) is used in a
wide antineoplastic spectrum. Doxorubicin exerts its cytotoxic
effect on tumor cells mainly by two mechanisms: (a) generation of
reactive oxygen species (ROS); and (b) inhibition of topoisomerase
II.
[0099] Doxorubicin is absorbed poorly and must be administered
intravenously. The pharmacokinetics are multicompartmental.
Distribution phases have half-lives of 12 min and 3.3 hr. The
elimination half-life is about 30 hr. Forty to 50% is secreted into
the bile. Most of the remainder is metabolized in the liver, partly
to an active metabolite (doxorubicinol), but a few percent is
excreted into the urine. In the presence of liver impairment, the
dose should be reduced.
[0100] Appropriate doses are, intravenous, adult, 60 to 75
mg/m.sup.2 at 21-day intervals or 25 to 30 mg/m.sup.2 on each of 2
or 3 successive days repeated at 3- or 4-wk intervals or 20
mg/m.sup.2 once a week. The lowest dose should be used in elderly
patients, when there is prior bone-marrow depression caused by
prior chemotherapy or neoplastic marrow invasion, or when the drug
is combined with other myelopoietic suppressant drugs. The dose
should be reduced by 50% if the serum bilirubin lies between 1.2
and 3 mg/dL and by 75% if above 3 mg/dL. The lifetime total dose
should not exceed 550 mg/m.sup.2 in patients with normal heart
function and 400 mg/m.sup.2 in persons having received mediastinal
irradiation. Children, 30 mg/m.sup.2 on each of 3 consecutive days,
repeated every 4 wk. Prescribing limits are as with adults.
[0101] b. Daunorubicin
[0102] Daunorubicin hydrochloride, 5,12-Naphthacenedione,
(8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexanopyrano-
syl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-10-methoxy-,
hydrochloride; also termed cerubidine and available from Wyeth.
Daunorubicin undergoes reduction to form oxygen free radical
intermediates. In the presence of oxygen and metal catalysts such
as Fe.sup.2+, daunorubicin undergoes reduction to the semiquinone
radical. In the presence of oxygen, the semiquinone radical can
form a superperoxide that in the presence of hydrogen peroxide
forms hydroxyl radicals.
[0103] In combination with other drugs it is included in the
first-choice chemotherapy of acute myelocytic leukemia in adults
(for induction of remission), acute lymphocytic leukemia and the
acute phase of chronic myelocytic leukemia. Oral absorption is
poor, and it must be given intravenously. The half-life of
distribution is 45 min and of elimination, about 19 hr. The
half-life of its active metabolite, daunorubicinol, is about 27 hr.
Daunorubicin is metabolized mostly in the liver and also secreted
into the bile (ca 40%). Dosage must be reduced in liver or renal
insufficiencies.
[0104] Suitable doses are (base equivalent), intravenous adult,
younger than 60 yr 45 mg/m2/day (30 mg/m2 for patients older than
60 yr) for 1, 2 or 3 days every 3 or 4 wk or 0.8 mg/kg/day for 3 to
6 days every 3 or 4 wk; no more than 550 mg/m2 should be given in a
lifetime, except only 450 mg/m.sup.2 ir there has been chest
irradiation; children, 25 mg/m2 once a week unless the age is less
than 2 yr or the body surface less than 0.5 m, in which case the
weight-based adult schedule is used. It is available in injectable
dosage forms (base equivalent) 20 mg (as the base equivalent to
21.4 mg of the hydrochloride).
[0105] C. Epirubicin
[0106] Epirubicin, (8
S-cis)-10-[(3-amino-2,3,6,-trideoxy-.alpha.-L-arabin-
o-hexopyranosyl)
oxyl]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyace-
tyl)-1-methoxy-5,12-naptha-cenedione (C.sub.27H.sub.29NO.sub.11),
is an anthracycline derived chemotherapeutic agent which is the
4'-epimer of doxorubicin and a semi-synthetic derivative of
daunorubicin. Epirubicin undergoes one-electron reduction to form
oxygen free radical intermediates. In the presence of oxygen and
metal catalysts such as Fe.sup.2+, epirubicin is reduced to the
semiquinone radical. In the presence of oxygen, the semiquinone
radical can form a superperoxide that in the presence of hydrogen
peroxide forms a hydroxyl radical.
[0107] Epirubcin is administered intravenously by infusion over
30-60 minutes rather than by direct injection. The drug is
extremely irritating to tissues and should thus not be administered
intramuscularly or subcutaneously.
[0108] 6. Arsenic Compounds
[0109] Arsenic agents have been used in ancient Chinese medicine
for several diseases. One of these agents, arsenic trioxide,
(As.sub.2O.sub.3), has recently been reported to be an effective
agent in therapy for relapse or refractory acute promyelocytic
leukemia. Data also suggests the therapeutic use of arsenic
trioxide for other hematologic cancers as well (Soignet et al.,
1999a; Wiemik et al., 1999, Geissler et al., 1999; Rousselot et
al., 1999). Organic arsinals, such as melarsoprol (Soignet et al.,
1999b) and an arsenic pyrimidine compound (U.S. Pat. No. 6,191,123)
may also have therapeutic implications.
[0110] 7. Retinoic Acid Derivatives
[0111] Retinoic acids such as all-trans retinoic acid (ATRA) or
9-cis retinoic acid are anticancer agent used in the clinical
treatment of cancer. It is a known regulator of cellular
proliferation and differentiation, and a known inhibitor of tumor
promotion. ATRA has been found to be especially useful in
hematological malignancies such as acute promyelocytic leukemia by
causing differentiation and apoptosis in immature malignant
promyelocytes. Doses of ATRA are normally about 45 mg/m2 per
day.
[0112] 8. Physical Agents that Generate Free Radicals
[0113] a. Heat (hyperthermia)
[0114] Hypothermia is a procedure in which a patient's tissue is
exposed to high temperatures (up to 106.degree. F.). External or
internal heating devices may be involved in the application of
local, regional, or whole-body hyperthermia. Local hyperthermia
involves the application of heat to a small area, such as a tumor.
Heat may be generated externally with high-frequency waves
targeting a tumor from a device outside the body. Internal heat may
involve a sterile probe, including thin, heated wires or hollow
tubes filled with warm water, implanted microwave antennae, or
radio-frequency electrodes. It is know that hyperthermia causes an
increased flux of free radicals in cells (Flanagan, 1998).
[0115] A patient's organ or a limb is heated for regional therapy,
which is accomplished using devices that produce high energy, such
as magnets. Alternatively, some of the patient's blood may be
removed and heated before being perfused into an area that will be
internally heated. Whole-body heating may also be implemented in
cases where cancer has spread throughout the body. Warm-water
blankets, hot wax, inductive coils, and thermal chambers may be
used for this purpose.
[0116] b. Ultraviolet Rays
[0117] Ultraviolet (UV) rays are invisible radiation with
wavelength of less than 400 nm. Ultraviolet rays are subdivided
into three regions, UVA (340-400 nm), UVB (290-320 run), and UVC
(200-290 nm). It is well known in the art that UV has various
effects on biological system, depending on the wavelength and the
intensity of the radiation. For example, ultraviolet at certain
range of wavelength causes damage to cellular DNA by formation of
pyrimidine dimers. The rate of induction of pyrimidine dimers is
maximal at 254 nm (Freeman et al, 1990). The UV-induced DNA damage
is believed to be a major mechanism responsible for the mutagenic
and carcinogenic effects of this type of radiation. At a sufficient
radiation intensity, UV causes cell death by induction of
apoptosis. It is also known in the art that UV rays, especially at
the wavelength of 280-400 nm, cause production of reactive oxygen
species. These free radicals subsequently damage cells and cause
apoptosis (Paretzoglou et al, 1998; Nishi et al, 1991; Nishigaki et
al, 1999). In the context of the instant invention, it is
contemplated that the use of 2-methoxyestradiol to inhibit SOD in
combination with a local ultraviolet radiation at the tumor site,
especially the tumor of skin, may cause a preferential accumulation
of free radicals in the cancer cells, and thus enhance the potency
and selectivity of cancer therapy.
[0118] C. X-rays and Gamma-rays
[0119] X-rays (roentgen rays) and Gamma-rays are two major types of
electromagnetic radiation wildly used in the medical field. In
atomic terms, X-rays are produced extranuclearly while gamma rays
are produced intranuclearly. The generation of electromagnetic
radiation and its used in medicine are well known in the art. When
electromagnetic radiation is applied to a biological system, the
energy of the radiation causes damage to cells by two possible
mechanisms. (1) The direct effect on important target molecules
such as DNA, which can be structurally damages and modified. (2)
production of free radicals by interaction of the energy rays with
water inside the cells or in the tissue matrix. The free radicals
then interact with important biological molecules such as DNA and
protein, and cause detrimental effects. In the context of the
instant invention, it is contemplated that the use of
2-methoxyestradiol to inhibit SOD in combination with a local
application of X-rays or gamma radiation at the tumor site may
cause a preferential accumulation of free radicals in the cancer
tissue, and thus enhance the potency and selectivity of cancer
therapy.
[0120] C. Combination Therapy
[0121] It is contemplated that the methods and compositions of the
instant invention will demonstrate effective tumoricidal
properties, nevertheless, it may be desirable, in certain
circumstances to combine the claimed therapeutic approach with
other cancer treatments. Thus, the claimed compositions may be
combined with other agents effective in the treatment of
hyperproliferative disease, such as other anti-cancer agents, or
with surgery. An "anti-cancer" agent is capable of negatively
affecting cancer in a subject, for example, by killing cancer
cells, inducing apoptosis in cancer cells, reducing the growth rate
of cancer cells, reducing the incidence or number of metastases,
reducing tumor size, inhibiting tumor growth, reducing the blood
supply to a tumor or cancer cells, promoting an immune response
against cancer cells or a tumor, preventing or inhibiting the
progression of cancer, or increasing the lifespan of a subject with
cancer. Anti-cancer agents include biological agents (biotherapy),
chemotherapy agents, and radiotherapy agents. More generally, these
other compositions would be provided in a combined amount effective
to kill or inhibit proliferation of the cell.
[0122] 1. Chemotherapy
[0123] Cancer therapies include a variety of combination therapies
with both chemical and radiation based treatments. Combination
chemotherapies include, for example, carboplatin, procarbazine,
mechlorethamine, cyclophosphamide, camptothecin, ifosfamide,
melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,
plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene,
estrogen receptor binding agents, taxol, gemcitabine, navelbine,
famesyl-protein transferase inhibitors, transplatinum,
5-fluorouracil, vincristine, vinblastine and methotrexate,
Temazolomide (an aqueous form of DTIC), or any analog or derivative
variant of the foregoing. In the context of the present invention,
it is contemplated that the disclosed methods and compositions
could be used in conjunction with one or more of the foregoing
chemotherapeutic agents. The combination of chemotherapy with
biological therapy is known as biochemotherapy.
[0124] 2. Gene Therapy
[0125] Tumor cell resistance to chemotherapy and radiotherapy
agents represents a major problem in clinical oncology. One goal of
current cancer research is to find ways to improve the efficacy of
chemotherapy, as for example, claimed in the instant invention, by
combining it with gene therapy. For example, the herpes
simplex-thymidine kinase (HS-tK) gene, when delivered to brain
tumors by a retroviral vector system, successfully induced
susceptibility to the antiviral agent ganciclovir (Culver et al.,
1992). In the context of the present invention, it is contemplated
that gene therapy could be used similarly in conjunction with the
disclosed methods and compositions.
[0126] Various combinations of therapies may be employed, the
claimed methods and compositions are "A" and the secondary agent,
such as radio- or chemotherapy, gene therapy or surgery, is
"B":
1 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A
B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B
B/A/A/A A/B/A/A A/A/B/A
[0127] It is expected that the treatment cycles would be repeated
as necessary. It also is contemplated that various standard
therapies, as well as surgical intervention, may be applied in
combination with the described therapy.
[0128] a. Inducers of Cellular Proliferation
[0129] The proteins that induce cellular proliferation further fall
into various categories dependent on function. The commonality of
all of these proteins is their ability to regulate cellular
proliferation and thus affect cellular metabolisms. For example,
the gene c-myc is known to stimulate cell proliferating activity
and cause an increase in production of free radical intermediates.
In one embodiment of the present invention, it is contemplated that
an ectopic overexpression of c-myc by gene transfer could be used
in conjunction with the disclosed methods and compositions to kill
cancer cells. As a second example, a form of PDGF, the sis
oncogene, is a secreted growth factor. Oncogenes rarely arise from
genes encoding growth factors, and at the present, sis is the only
known naturally-occurring oncogenic growth factor. In one
embodiment of the present invention, it is contemplated that
anti-sense mRNA directed to a particular inducer of cellular
proliferation is used to prevent expression of the inducer of
cellular proliferation.
[0130] The proteins FMS, ErbA, ErbB and neu are growth factor
receptors. Mutations to these receptors result in loss of
regulatable function. For example, a point mutation affecting the
transmembrane domain of the Neu receptor protein results in the neu
oncogene. The erbA oncogene is derived from the intracellular
receptor for thyroid hormone. The modified oncogenic ErbA receptor
is believed to compete with the endogenous thyroid hormone
receptor, causing uncontrolled growth.
[0131] The largest class of oncogenes includes the signal
transducing proteins (e.g., Src, Abl and Ras). The protein Src is a
cytoplasmic protein-tyrosine kinase, and its transformation from
proto-oncogene to oncogene in some cases, results via mutations at
tyrosine residue 527. In contrast, transformation of GTPase protein
ras from proto-oncogene to oncogene, in one example, results from a
valine to glycine mutation at amino acid 12 in the sequence,
reducing ras GTPase activity.
[0132] The proteins Jun, Fos and Myc are proteins that directly
exert their effects on nuclear functions as transcription
factors.
[0133] b. Inhibitors of Cellular Proliferation
[0134] The tumor suppressor genes function to inhibit excessive
cellular proliferation. The inactivation of these genes destroys
their inhibitory activity, resulting in unregulated proliferation.
The tumor suppressors p53, p16 and C-CAM are described below.
[0135] High levels of mutant p53 have been found in many cells
transformed by chemical carcinogenesis, ultraviolet radiation, and
several viruses. The p53 gene is a frequent target of mutational
inactivation in a wide variety of human tumors and is already
documented to be the most frequently mutated gene in common human
cancers. It is mutated in over 50% of human NSCLC (Hollstein et
al., 1991) and in a wide spectrum of other tumors.
[0136] The p53 gene encodes a 393-amino acid phosphoprotein that
can form complexes with host proteins such as large-T antigen and
E1B. The protein is found in normal tissues and cells, but at
concentrations which are minute by comparison with transformed
cells or tumor tissue
[0137] Wild-type p53 is recognized as an important growth regulator
in many cell types. Missense mutations are common for the p53 gene
and are essential for the transforming ability of the oncogene. A
single genetic change prompted by point mutations can create
carcinogenic p53. Unlike other oncogenes, however, p53 point
mutations are known to occur in at least 30 distinct codons, often
creating dominant alleles that produce shifts in cell phenotype
without a reduction to homozygosity. Additionally, many of these
dominant negative alleles appear to be tolerated in the organism
and passed on in the germ line. Various mutant alleles appear to
range from minimally dysfunctional to strongly penetrant, dominant
negative alleles (Weinberg, 1991).
[0138] Another inhibitor of cellular proliferation is p16. The
major transitions of the eukaryotic cell cycle are triggered by
cyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent
kinase 4 (CDK4), regulates progression through the G.sub.1. The
activity of this enzyme may be to phosphorylate Rb at late G.sub.1.
The activity of CDK4 is controlled by an activating subunit, D-type
cyclin, and by an inhibitory subunit, the p16.sup.INK4 has been
biochemically characterized as a protein that specifically binds to
and inhibits CDK4, and thus may regulate Rb phosphorylation
(Serrano et al., 1993; Serrano et al., 1995). Since the
p16.sup.INK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion
of this gene may increase the activity of CDK4, resulting in
hyperphosphorylation of the Rb protein. p16.sup.INK4 belongs to a
newly described class of CDK-inhibitory proteins that also includes
p.sub.16.sup.B, p19, p21.sup.WAF1, and p.sub.27.sup.KIP1. The
p16.sup.INK4 gene maps to 9p21, a chromosome region frequently
deleted in many tumor types. Homozygous deletions and mutations of
the p16.sup.INK4 gene are frequent in human tumor cell lines. Other
genes that may be employed according to the present invention
include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zacl, p73,
VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27
fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F,
ras, myc, neu, raf, erb, fins, trk, ret, gsp, hst, abl, E1A, p300,
genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin,
BAI-1, GDAIF, or their receptors) and MCC.
[0139] C. Regulators of Programmed Cell Death
[0140] Apoptosis, or programmed cell death, is an essential process
for normal embryonic development, maintaining homeostasis in adult
tissues, and suppressing carcinogenesis (Kerr et al., 1972). The
Bcl-2 family of proteins and ICE-like proteases have been
demonstrated to be important regulators and effectors of apoptosis
in other systems. The Bcl-2 protein, discovered in association with
follicular lymphoma, plays a prominent role in controlling
apoptosis and enhancing cell survival in response to diverse
apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985;
Cleary et al, 1986; Tsujimoto et al., 1985; Tsujimoto and Croce,
1986). The evolutionarily conserved Bcl-2 protein now is recognized
to be a member of a family of related proteins, which can be
categorized as death agonists or death antagonists.
[0141] Subsequent to its discovery, it was shown that Bcl-2 acts to
suppress cell death triggered by a variety of stimuli. Also, it now
is apparent that there is a family of Bcl-2 cell death regulatory
proteins which share in common structural and sequence homologies.
These different family members have been shown to either possess
similar functions to Bcl-2 (e.g., Bcl.sub.XL, Bcl.sub.W, BCl.sub.S,
MCl-1, Al, Bfl-1) or counteract Bcl-2 function and promote cell
death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
[0142] 3. Radiotherapy
[0143] Other factors that cause DNA damage and have been used
extensively include what are commonly known as .gamma.-rays,
X-rays, and/or the directed delivery of radioisotopes to tumor
cells. Other forms of DNA damaging factors are also contemplated
such as microwaves and UV-irradiation. It is most likely that all
of these factors effect a broad range of damage on DNA, on the
precursors of DNA, on the replication and repair of DNA, and on the
assembly and maintenance of chromosomes. Dosage ranges for X-rays
range from daily doses of 50 to 200 roentgens for prolonged periods
of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
Dosage ranges for radioisotopes vary widely, and depend on the
half-life of the isotope, the strength and type of radiation
emitted, and the uptake by the neoplastic cells.
[0144] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a therapeutic
construct and a chemotherapeutic or radiotherapeutic agent are
delivered to a target cell or are placed in direct juxtaposition
with the target cell. To achieve cell killing or stasis, both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing.
[0145] 4. Immunotherapy
[0146] Immunotherapeutics, generally, rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
The immune effector may be, for example, an antibody specific for
some marker on the surface of a tumor cell. The antibody alone may
serve as an effector of therapy or it may recruit other cells to
actually effect cell killing. The antibody also may be conjugated
to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain,
cholera toxin, pertussis toxin, etc.) and serve merely as a
targeting agent. Alternatively, the effector may be a lymphocyte
carrying a surface molecule that interacts, either directly or
indirectly, with a tumor cell target. Various effector cells
include cytotoxic T cells and NK cells.
[0147] Immunotherapy may be useful as part of a combined therapy.
In one aspect of immunotherapy, the tumor cell must bear some
marker that is amenable to targeting, i.e., is not present on the
majority of other cells. Many tumor markers exist and any of these
may be suitable for targeting in the context of the present
invention. Common tumor markers include carcinoembryonic antigen,
prostate specific antigen, urinary tumor associated antigen, fetal
antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis
Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb
B and p155.
[0148] a. Passive Immunotherapy
[0149] A number of different approaches for passive immunotherapy
of cancer exist. They may be broadly categorized into the
following: injection of antibodies alone; injection of antibodies
coupled to toxins or chemotherapeutic agents; injection of
antibodies coupled to radioactive isotopes; injection of
anti-idiotype antibodies; and finally, purging of tumor cells in
bone marrow.
[0150] Preferably, human monoclonal antibodies are employed in
passive immunotherapy, as they produce few or no side effects in
the patient. However, their application is somewhat limited by
their scarcity and have so far only been administered
intralesionally. Human monoclonal antibodies to ganglioside
antigens have been administered intralesionally to patients
suffering from cutaneous recurrent melanoma (Irie & Morton,
1986). Regression was observed in six out of ten patients,
following, daily or weekly, intralesional injections. In another
study, moderate success was achieved from intralesional injections
of two human monoclonal antibodies (Irie et al., 1989).
[0151] It may be favorable to administer more than one monoclonal
antibody directed against two different antigens or even antibodies
with multiple antigen specificity. Treatment protocols also may
include administration of lymphokines or other immune enhancers as
described by Bajorin et al., (1988). The development of human
monoclonal antibodies is described in further detail elsewhere in
the specification.
[0152] b. Active Immunotherapy
[0153] In active immunotherapy, an antigenic peptide, polypeptide
or protein, or an autologous or allogenic tumor cell composition or
"vaccine" is administered, generally with a distinct bacterial
adjuvant (Ravindranath & Morton, 1991; Morton &
Ravindranath, 1996; Morton et al., 1992; Mitchell et al., 1990;
Mitchell et al., 1993). In melanoma immunotherapy, those patients
who elicit high IgM response often survive better than those who
elicit no or low IgM antibodies (Morton et al., 1992). IgM
antibodies are often transient antibodies and the exception to the
rule appears to be anti-ganglioside or anticarbohydrate
antibodies.
[0154] C. Adoptive Immunotherapy
[0155] In adoptive immunotherapy, the patient's circulating
lymphocytes, or tumor infiltrated lymphocytes, are isolated in
vitro, activated by lymphokines such as IL-2 or transduced with
genes for tumor necrosis, and readministered (Rosenberg et al.,
1988; 1989). To achieve this, one would administer to an animal, or
human patient, an immunologically effective amount of activated
lymphocytes in combination with an adjuvant-incorporated anigenic
peptide composition as described herein. The activated lymphocytes
will most preferably be the patient's own cells that were earlier
isolated from a blood or tumor sample and activated (or "expanded")
in vitro. This form of immunotherapy has produced several cases of
regression of melanoma and renal carcinoma, but the percentage of
responders were few compared to those who did not respond.
[0156] 5. Surgery
[0157] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative and palliative surgery. Curative surgery is a
cancer treatment that may be used in conjunction with other
therapies, such as the treatment of the present invention,
chemotherapy, radiotherapy, hormonal therapy, gene therapy,
immunotherapy and/or alternative therapies.
[0158] Curative surgery includes resection in which all or part of
cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
miscopically controlled surgery (Mohs' surgery). It is further
contemplated that the present invention may be used in conjunction
with removal of superficial cancers, precancers, or incidental
amounts of normal tissue.
[0159] Upon excision of part of all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well.
[0160] 6. Other agents
[0161] It is contemplated that other agents may be used in
combination with the present invention to improve the therapeutic
efficacy of treatment. These additional agents include
immunomodulatory agents, agents that affect the upregulation of
cell surface receptors and GAP junctions, cytostatic and
differentiation agents, inhibitors of cell adehesion, agents that
increase the sensitivity of the hyperproliferative cells to
apoptotic inducers, or other biological agents. Immunomodulatory
agents include tumor necrosis factor; interferon alpha, beta, and
gamma; IL-2 and other cytokines; F42K and other cytokine analogs;
or MIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines. It is
further contemplated that the upregulation of cell surface
receptors or their ligands such as Fas/Fas ligand, DR4 or DR5/TRAIL
would potentiate the apoptotic inducing abililties of the present
invention by establishment of an autocrine or paracrine effect on
hyperproliferative cells. Increases intercellular signaling by
elevating the number of GAP junctions would increase the
anti-hyperproliferative effects on the neighboring
hyperproliferative cell population. In other embodiments,
cytostatic or differentiation agents can be used in combination
with the present invention to improve the anti-hyerproliferative
efficacy of the treatments. Inhibitors of cell adhesion are
contemplated to improve the efficacy of the present invention.
Examples of cell adhesion inhibitors are focal adhesion kinase
(FAKs) inhibitors and Lovastatin. It is further contemplated that
other agents that increase the sensitivity of a hyperproliferative
cell to apoptosis, such as the antibody c225, could be used in
combination with the present invention to improve the treatment
efficacy.
[0162] Hormonal therapy may also be used in conjunction with the
present invention or in combination with any other cancer therapy
previously described. The use of hormones may be employed in the
treatment of certain cancers such as breast, prostate, ovarian, or
cervical cancer to lower the level or block the effects of certain
hormones such as testosterone or estrogen. This treatment is often
used in combination with at least one other cancer therapy as a
treatment option or to reduce the risk of metastases.
[0163] D. Pharmaceutical Compositions
[0164] 1. Pharmaceutically Acceptable Carriers
[0165] Aqueous compositions of the present invention comprise an
effective amount of 2-methoxyestradiol dissolved and/or dispersed
in a pharmaceutically acceptable carrier and/or aqueous medium and
an effective amount of a compound that increases intracellular
O.sub.2.sup.- concentration dissolved and/or dispersed in a
pharmaceutically acceptable carrier and/or aqueous medium. The
phrases "pharmaceutically and/or pharmacologically acceptable"
refer to molecular entities and/or compositions that do not produce
an adverse, allergic and/or other untoward reaction when
administered to an animal, and specifically to humans, as
appropriate.
[0166] As used herein, "pharmaceutically acceptable carrier"
includes any and/or all solvents, dispersion media, coatings,
antibacterial and/or antifungal agents, isotonic and/or absorption
delaying agents and/or the like. The use of such media and/or
agents for pharmaceutical active substances is well known in the
art. Except insofar as any conventional media and/or agent is
incompatible with the active ingredient, its use in the therapeutic
compositions is contemplated. Supplementary active ingredients can
also be incorporated into the compositions. For administration to
humans, preparations should meet sterility, pyrogenicity, general
safety and/or purity standards as required by FDA Office of
Biologics standards.
[0167] The 2-methoxyestradiol and a compound that increases
intracellular O.sub.2.sup.- concentration should be extensively
purified to remove undesired small molecular weight molecules
and/or lyophilized for more ready formulation into a desired
vehicle, where appropriate. The active compounds will then
generally be formulated for oral administration, or for parenteral
administration, e.g., formulated for injection via the intravenous,
intramuscular, sub-cutaneous, intralesional, and/or even
intraperitoneal routes. The preparation of an aqueous composition
that contains a 2-methoxyestradiol agent and/or the compound that
increases intracellular O.sub.2.sup.- concentration as an active
component and/or ingredient will be known to those of skill in the
art in light of the present disclosure. Typically, such
compositions can be prepared as injectables, either as liquid
solutions and/or suspensions; solid forms suitable for using to
prepare solutions and/or suspensions upon the addition of a liquid
prior to injection can also be prepared; and/or the preparations
can also be emulsified.
[0168] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions and/or dispersions; formulations
including sesame oil, peanut oil and/or aqueous propylene glycol;
and/or sterile powders for the extemporaneous preparation of
sterile injectable solutions and/or dispersions. In all cases the
form must be sterile and/or must be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and/or storage and/or must be preserved against the
contaminating action of microorganisms, such as bacteria and/or
fungi.
[0169] Solutions of the active compounds as free base and/or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and/or mixtures thereof and/or in oils. Under ordinary
conditions of storage and/or use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0170] The 2-methoxyestradiol and a compound that increases
intracellular O.sub.2.sup.- concentration can be formulated into a
composition in a neutral and/or salt form. Pharmaceutically
acceptable salts, include the acid addition salts (formed with the
free amino groups of the protein) and/or which are formed with
inorganic acids such as, for example, hydrochloric and/or
phosphoric acids, and/or such organic acids as acetic, oxalic,
tartaric, mandelic, and/or the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, and/or ferric
hydroxides, and/or such organic bases as isopropylamine,
trimethylamine, histidine, procaine and/or the like. In terms of
using peptide therapeutics as active ingredients, the technology of
U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230;
4,596,792; and/or 4,578,770, each incorporated herein by reference,
may be used.
[0171] The carrier can also be a solvent and/or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and/or liquid polyethylene glycol,
and/or the like), suitable mixtures thereof, and/or vegetable oils.
The proper fluidity can be maintained, for example, by the use of a
coating, such as lecithin, by the maintenance of the required
particle size in the case of dispersion and/or by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial and/or antifungal agents,
for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal, and/or the like. In many cases, it will be preferable
to include isotonic agents, for example, sugars and/or sodium
chloride. Prolonged absorption of the injectable compositions can
be brought about by the use in the compositions of agents delaying
absorption, for example, aluminum monostearate and/or gelatin.
[0172] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and/or the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and/or freeze-drying techniques
which yield a powder of the active ingredient plus any additional
desired ingredient from a previously sterile-filtered solution
thereof. The preparation of more, and/or highly, concentrated
solutions for direct injection is also contemplated, where the use
of DMSO as solvent is envisioned to result in extremely rapid
penetration, delivering high concentrations of the active agents to
a small tumor area.
[0173] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and/or in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and/or the
like can also be employed.
[0174] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary
and/or the liquid diluent first rendered isotonic with sufficient
saline and/or glucose. These particular aqueous solutions are
especially suitable for intravenous, intramuscular, subcutaneous
and/or intraperitoneal administration. In this connection, sterile
aqueous media which can be employed will be known to those of skill
in the art in light of the present disclosure. For example, one
dosage could be dissolved in 1 ml of isotonic NaCl solution and/or
either added to 1000 ml of hypodermoclysis fluid and/or injected at
the proposed site of infusion, (see for example, "Remington's
Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and/or
1570-1580). Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject.
[0175] 2-methoxyestradiol and a compound that increases
intracellular O.sub.2.sup.- concentration may be formulated within
a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams,
and/or about 0.001 to 0.1 milligrams, and/or about 0.1 to 1.0
and/or even about 10 milligrams per dose and/or so. Multiple doses
can also be administered.
[0176] In addition to the compounds formulated for parenteral
administration, such as intravenous and/or intramuscular injection,
other pharmaceutically acceptable forms include, e.g., tablets
and/or other solids for oral administration; liposomal
formulations; time release capsules; and/or any other form
currently used, including cremes.
[0177] One may also use nasal solutions and/or sprays, aerosols
and/or inhalants in the present invention. Nasal solutions are
usually aqueous solutions designed to be administered to the nasal
passages in drops and/or sprays. Nasal solutions are prepared so
that they are similar in many respects to nasal secretions, so that
normal ciliary action is maintained. Thus, the aqueous nasal
solutions usually are isotonic and/or slightly buffered to maintain
a pH of 5.5 to 6.5. In addition, antimicrobial preservatives,
similar to those used in ophthalmic preparations, and/or
appropriate drug stabilizers, if required, may be included in the
formulation. Various commercial nasal preparations are known and/or
include, for example, antibiotics and/or antihistamines and/or are
used for asthma prophylaxis.
[0178] Additional formulations which are suitable for other modes
of administration include vaginal suppositories and/or pessaries. A
rectal pessary and/or suppository may also be used. Suppositories
are solid dosage forms of various weights and/or shapes, usually
medicated, for insertion into the rectum, vagina and/or the
urethra. After insertion, suppositories soften, melt and/or
dissolve in the cavity fluids. In general, for suppositories,
traditional binders and/or carriers may include, for example,
polyalkylene glycols and/or triglycerides; such suppositories may
be formed from mixtures containing the active ingredient in the
range of 0.5% to 10%, preferably 1%-2%.
[0179] Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and/or the like. These compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations and/or powders. In certain defined embodiments, oral
pharmaceutical compositions will comprise an inert diluent and/or
assimilable edible carrier, and/or they may be enclosed in hard
and/or soft shell gelatin capsule, and/or they may be compressed
into tablets, and/or they may be incorporated directly with the
food of the diet. For oral therapeutic administration, the active
compounds may be incorporated with excipients and/or used in the
form of ingestible tablets, buccal tables, troches, capsules,
elixirs, suspensions, syrups, wafers, and/or the like. Such
compositions and/or preparations should contain at least 0.1% of
active compound. The percentage of the compositions and/or
preparations may, of course, be varied and/or may conveniently be
between about 2 to about 75% of the weight of the unit, and/or
preferably between 25-60%. The amount of active compounds in such
therapeutically useful compositions is such that a suitable dosage
will be obtained.
[0180] The tablets, troches, pills, capsules and/or the like may
also contain the following: a binder, as gum tragacanth, acacia,
cornstarch, and/or gelatin; excipients, such as dicalcium
phosphate; a disintegrating agent, such as corn starch, potato
starch, alginic acid and/or the like; a lubricant, such as
magnesium stearate; and/or a sweetening agent, such as sucrose,
lactose and/or saccharin may be added and/or a flavoring agent,
such as peppermint, oil of wintergreen, and/or cherry flavoring.
When the dosage unit form is a capsule, it may contain, in addition
to materials of the above type, a liquid carrier. Various other
materials may be present as coatings and/or to otherwise modify the
physical form of the dosage unit. For instance, tablets, pills,
and/or capsules may be coated with shellac, sugar and/or both. A
syrup of elixir may contain the active compounds sucrose as a
sweetening agent methyl and/or propylparabens as preservatives, a
dye and/or flavoring, such as cherry and/or orange flavor.
[0181] 2. Liposomes and/or Nanocapsules
[0182] In certain embodiments, the use of liposomes and/or
nanoparticles is contemplated for the introduction of
2-methoxyestradiol and the compound the increases intracellular
O.sub.2.sup.- concentration into host cells. The formation and/or
use of liposomes is generally known to those of skill in the art,
and/or is also described below.
[0183] Nanocapsules can generally entrap compounds in a stable
and/or reproducible way. To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) should be designed using polymers able to be degraded in
vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet
these requirements are contemplated for use in the present
invention, and/or such particles may be are easily made.
[0184] Liposomes are formed from phospholipids that are dispersed
in an aqueous medium and/or spontaneously form multilamellar
concentric bilayer vesicles (also termed multilamellar vesicles
(MLVs). MLVs generally have diameters of from 25 nm to 4 .mu.m.
Sonication of MLVs results in the formation of small unilamellar
vesicles (SUVs) with diameters in the range of 200 to 500 .ANG.,
containing an aqueous solution in the core.
[0185] The following information may also be utilized in generating
liposomal formulations. Phospholipids can form a variety of
structures other than liposomes when dispersed in water, depending
on the molar ratio of lipid to water. At low ratios the liposome is
the preferred structure. The physical characteristics of liposomes
depend on pH, ionic strength and/or the presence of divalent
cations. Liposomes can show low permeability to ionic and/or polar
substances, but at elevated temperatures undergo a phase transition
which markedly alters their permeability. The phase transition
involves a change from a closely packed, ordered structure, known
as the gel state, to a loosely packed, less-ordered structure,
known as the fluid state. This occurs at a characteristic
phase-transition temperature and/or results in an increase in
permeability to ions, sugars and/or drugs.
[0186] Liposomes interact with cells via four different mechanisms:
Endocytosis by phagocytic cells of the reticuloendothelial system
such as macrophages and/or neutrophils; adsorption to the cell
surface, either by nonspecific weak hydrophobic and/or
electrostatic forces, and/or by specific interactions with
cell-surface components; fusion with the plasma cell membrane by
insertion of the lipid bilayer of the liposome into the plasma
membrane, with simultaneous release of liposomal contents into the
cytoplasm; and/or by transfer of liposomal lipids to cellular
and/or subcellular membranes, and/or vice versa, without any
association of the liposome contents. Varying the liposome
formulation can alter which mechanism is operative, although more
than one may operate at the same time.
[0187] E. Kits
[0188] Therapeutic kits of the present invention are kits
comprising 2-methoxyestradiol and a compound that increases
intracellular O.sub.2.sup.- concentration. Such kits will generally
contain, in suitable container means, and a pharmaceutically
acceptable formulation of 2-methoxyestradiol a compound that
increases intracellular O.sub.2.sup.-. The kit may have a single
container means, and/or it may have distinct container means for
each compound.
[0189] When the components of the kit are provided in one and/or
more liquid solutions, the liquid solution is an aqueous solution,
with a sterile aqueous solution being particularly preferred.
2-methoxyestradiol compositions and compositions of a compound that
increases intracellular O.sub.2.sup.- concentration may also be
formulated into a syringeable composition. In which case, the
container means may itself be a syringe, pipette, and/or other such
like apparatus, from which the formulation may be applied to an
infected area of the body, injected into an animal, and/or even
applied to and/or mixed with the other components of the kit.
[0190] However, the components of the kit may be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means.
[0191] The components of the kit may be provided as solid tablets
or capsules containing 2-methoxyestradiol and tablets or capsules
containing a compound that increases intracellular O.sub.2.sup.-
concentration for oral administration, either simultaneously or
sequentially with the tablets/capsules containing each
component.
[0192] The container means will generally include at least one
vial, test tube, flask, bottle, syringe and/or other container
means, into which the 2-methoxyestradiol and a compound that
increases intracellular O.sub.2.sup.- concentration are placed,
preferably, suitably allocated. The kits may also comprise a second
container means for containing a sterile, pharmaceutically
acceptable buffer and/or other diluent.
[0193] The kits of the present invention will also typically
include a means for containing the vials in close confinement for
commercial sale, such as, e.g., injection and/or blow-molded
plastic containers into which the desired vials are retained.
[0194] Irrespective of the number and/or type of containers, the
kits of the invention may also comprise, and/or be packaged with,
an instrument for assisting with the injection/administration
and/or placement of the 2-methoxyestradiol composition and a
composition with a compound that increases intracellular
O.sub.2.sup.- concentration within the body of an animal. Such an
instrument may be a syringe, pipette, forceps, and/or any such
medically approved delivery vehicle.
EXAMPLES
[0195] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
[0196] Methods
[0197] Assay of SOD Activity
[0198] A spectrophotometric assay was used to determine the effect
of 2-methoxyestradiol (2-ME) on SOD activity in vitro with purified
enzymes (bovine CuZnSOD from Boehringer Mannheim; human CuZnSOD and
E. coli MnSOD from Sigma). The reactions contained 2.5 ml of 50 mM
Na.sub.2CO.sub.3, 0.1 ml of 3 mM xanthine, 0.1 ml of 3 mM EDTA, and
0.1 ml of 0.8 mM XTT
(3'-(1-[phenylamino-carbonyl]3,4-tetrazolium)-bis(4-methoxy-6-nitro)benze-
ne-sulfonic acid hydrate), and the indicated concentrations of SOD
and 2-ME. Xanthine oxidase (0.1 ml, 64 mU/ml) was added to start
the reaction. After incubation at 24.degree. C. for 30 min, the
absorbance at 470 nm was measured. The relative activity (RA) of
SOD was calculated by the following formula:
RA=[1-(c-b)/(a-b)].times.100%
[0199] where a is the absorbance of reaction without SOD, b is the
absorbance of reaction with SOD but without 2-ME, c is the
absorbance of reaction with SOD in the presence of 2-ME. All values
were subtracted by the background reading of the blank. In separate
experiments using nitro blue tetrazolium or cytochrome c as
substrates for SOD assay, 2-ME and DMSO (as a solvent for 2-ME)
interfered with the calorimetric reaction. These artifacts preclude
the use of NBT or cytochrome c as the substrates for measuring the
effect of 2-ME on SOD activity. Neither 2-ME nor DMSO affect the
calorimetric reaction of XTT.
[0200] Quantitation of Cellular O.sub.2.sup.- and H.sub.2O.sub.2
Production
[0201] The method using hydroethidine to detect O.sub.2.sup.- in
tissue sections was adapted for quantitation of intracellular
O.sub.2.sup.- by flow cytometry analysis. This assay is based on
the unique chemical properties of hydroethidine, a weak blue
fluorescent dye which is selectively converted by O.sub.2.sup.- to
ethidium with a bright red fluorescence. The control and
drug-treated cells (1.5.times.10.sup.6/samp- le) were incubated
with hydroethidine (20 ng/ml) for 60 min, washed twice with 3 ml
PBS, resuspended in 2 ml PBS, and then analyzed within 1 h by flow
cytometry, using the red laser channel. A microassay was used to
measure the effect of 2-ME on H.sub.2O.sub.2 generation in cell
culture. Exponentially growing cells were harvested, suspended at
the density of 10.sup.7/ml, and incubated with 2-ME in a modified
KRPG solution containing 145 mM NaCl, 5.7 mM Na.sub.2HPO.sub.4 (pH
7.35), 4.86 mM KCl, 0.54 mM CaCl.sub.2, 1.22 mM MgSO.sub.4,
horseradish peroxidase (type II, 1 unit/ml), and 50 .mu.M
N-acetyl-3,7-dihydroxyphenoxazine (A6550). The horseradish
peroxidase converted A6550 (non-fluorescent) to a highly
fluorescent product in the presence of H.sub.2O.sub.2. The
intensity of the fluorescence was linearly proportional to
H.sub.2O.sub.2 as long as the ratio of A6550:H.sub.2O.sub.2 was
greater than 5 in the reaction mixtures. Samples were incubated in
a 96-well plate for 2 h and then read at 590/645 nm
(excitation/emission wavelengths). Various concentrations of pure
H.sub.2O.sub.2 were used in parallel reactions to construct a
standard curve.
[0202] Assay of Gene Expression by cDNA Microarray Analysis
[0203] The Atlas.TM. Human cDNA Expression Array I and the
Atlas.TM. Human Cancer cDNA Expression Array (ClonTech
Laboratories, Inc.) were used in this study. One microgram of mRNA
isolated from the control or drug-treated cells was converted to
radioactive cDNA by reverse transcription in the presence of
[.alpha.-.sup.32P]-dATP. The .sup.32P-labeled cDNA was then
denatured and hybridized to the eDNA expression arrays according to
the procedures recommended by the manufacturer. The radioactivity
on the membranes was quantified by a phosphoimager. Change in gene
expression after 2-ME treatment was calculated as percentage of the
untreated cells, using three of the internal controls (ubiquitin,
GAPDH, and the 23 kD highly basic protein) recommended by the
manufacturer for normalization to ensure the comparability of the
control and drug-treated samples.
[0204] RT-PCR Analysis
[0205] The expression of SOD RNA was also measured by RT-PCR. RNA
(1 .mu.g) isolated from the control or 2-ME-treated cells was first
converted to cDNA by reverse transcription using SuperScript II
reverse transcriptase (GibcoBRL) and the anchored oligo-dT primer
set (Genosys). The cDNA was than amplified by PCR. The SOD1primers
were from Genosys (forward, SEQ ID NO: 3,
5'-ACGAAGGCCGTGTGCGTGCTGAA; backward, SEQ ID NO: 4,
5'-ACCACAAGCCAAACGACTTCCAGC). The reaction was run at 94.degree. C.
(1 min).fwdarw.60.degree. C. (1 min).fwdarw.72.degree. C. (1 min)
for 20 cycles, which was within the linear reaction window. GAPDH
was also measured by RT-PCR from the same RNA samples and used as
an internal control (GAPDH primers: forward, SEQ ID NO: 5,
5'-CCATCAATGACCCCTTCA TTGACC; backward, SEQ ID NO: 6,
5'-GAAGGCCATGCCAGTGAGCTTCC).
[0206] Assays of Cellular Accumulation and Metabolism of 2-ME
[0207] To determine cellular uptake of 2-ME, cells were incubated
with 1 .mu.M [.sup.3H]2-ME (0.5 .mu.Ci/ml) for 5 h and when washed
twice with cold PBS. Radioactivity associated with the cell pellets
or the culture medium was quantitated by liquid scintillation
counting. 2-ME concentrations were calculated based on the specific
radioactivity of [.sup.3H]2-ME, cell number, and the mean cell
volume as measured by a Coulter Counter equipped with a
256-Channelyzer. HPLC was used to analyze potential cellular
metabolites of 2-ME. Cells were incubated with [.sup.3H]2-ME (0.5
.mu.Ci/ml) for 5 h, washed twice with PBS, and then extracted with
50% methanol. The extracts were analyzed by HPLC equipped with a UV
detector (284 nm) and an on-line liquid scintillation counter,
using a .mu.Bondapak C.sub.18 reversed-phase column and the
following running conditions: 1 ml/min; 0-5 min, 100% buffer A
(water:acetontrile:acetic acid=59:40:1); 5-17 min, a linear
gradient of 0->100% buffer B (water:acetontrile:acetic
acid=39:60:1); 17-30 min, 100% B.
[0208] Results
[0209] 2-ME was originally employed to increase p53 protein in an
attempt to enhance cellular response to other anticancer agents.
Surprisingly, 2-ME itself showed a potent activity against human
leukemia cells with different p53 genotypes. Typical apoptotic
morphology and nucleosomal DNA fragmentation were observed in ML-1
(wt p53) and HL-60 (p53.sup.-) cells treated with 1 .mu.M 2-ME
(FIG. 17A). The MTT assay further demonstrated that cells with wt
p53 (ML-1), mutant p53 (CEM and Raji), and no p53 (K562 and HL-60)
were similarly sensitive to 2-ME (FIGS. 8-10). The IC.sub.50 was
less than 1 .mu.M for all five cell lines.
[0210] When normal lymphocytes from 8 healthy donors were incubated
with 2-ME, no apparent loss of cell survival was observed (FIGS.
35-36), suggesting that 2-ME may have a selective anti-leukemia
activity. Because normal lymphocytes were quiescent and might not
be comparable to proliferating leukemia cells, we further used
quiescent leukemia cells isolated from patients with chronic
lymphocytic leukemia (CLL) for comparison with normal lymphocytes.
As shown in FIGS. 6-8, 2-ME caused a substantial loss of cell
survival in CLL cells. This compound also induced apoptosis in
leukemia cells from a CML (chronic myelogenous leukemia) patient in
relapsed blast crisis (FIG. 5e, lane 3), that were resistant to
ara-C in vitro (lane 2). Evaluation of the activity of 2-ME in
primary leukemia cells from a total of 31 CLL patients, 3 CML
patients, 6 AML (acute myelogenous leukemia) patients, and 2 ALL
(acute lymphocytic leukemia) patients revealed a substantial
activity in the majority of the cases, although the degree of
cytotoxicity varied among samples (FIG. 17D). Overall, 30 .mu.M
2-ME reduced cell survival to 32.7% in CLL cells, 54.7% in CML
cells, 49.6% in AML cells, and 56.5% in ALL cells. 2-ME was
significantly more toxic to CLL cells than to normal lymphocytes at
both 10 and 30 .mu.M (p<0.0001, FIG. 17E).
[0211] To test effect of 2-ME in the normal lymphocytes induced to
re-enter the proliferating cell cycle, lymphocytes were first
stimulated with phytohemagglutinin (PHA-M) and then exposed to
2-ME. PHA (5 .mu.g/ml, 48 h) induced significant proliferative
activity, as measured by [.sup.3H]thymidine incorporation (6,903
dpm->99,875 dpm) and by flow cytometry analysis (S phase:
0%->17.1%; G2/M phases: 0%->9.5%; n--3). When the lymphocytes
were incubated with 2-ME (0-30 .mu.M) for an additional 3 days, no
nucleosomal DNA fragmentation was detected (FIG. 17B, lanes 1-4). A
small amount of DNA smearing was seen in all PHA-stimulated cells
(lanes 5-8), suggesting some spontaneous turnover of the cells
during the 5-day incubation. MTT assay revealed that 2-ME inhibited
the proliferation of the PHA-treated lymphocytes, which
nevertheless remained viable during the 3-day drug incubation (FIG.
17C).
[0212] The cDNA microarray assay (Atlas Human cDNA Expression
Array, ClonTech) was used to search for candidate molecules
involved in the cellular responses to 2-ME. As illustrated in FIG.
11, the gene located in the second row of the last column (in
doublets.) showed a significant increase of mRNA expression 5 h
after 2-ME incubation. This molecule was identified as CuZnSOD
(SOD1, GenBank accession code, k00065). Quantitation by
phosphoimager revealed that expression of CuZnSOD in 2-ME-treated
cells was 236% of the control, after the signal was normalized by
three internal standards (see Methods). This increase was confirmed
by a RT-PCR assay (FIG. 18A). Similar increase of CuZnSOD mRNA
expression was also observed in HL-60 cells.
[0213] To further investigate the mechanism responsible for the
increase in SOD mRNA expression, the following two possibilities
were tested: (1) 2-ME might cause an increase of cellular
O.sub.2.sup.- production and thus induce SOD expression in response
to the free radical stress. This would lead to an increase of
H.sub.2O.sub.2 production. (2) 2-ME might inhibit SOD enzyme
activity and cause a feedback upregulation of SOD expression. This
would predict a decrease in H.sub.2O.sub.2 generation. In fact,
2-ME caused a concentration-dependent decrease of H.sub.2O.sub.2 in
ML-1 and HL-60 cells (FIG. 13). Immunoblot analysis showed that the
reduced H.sub.2O.sub.2 production was not due to a loss of CuZnSOD
or MnSOD protein (FIG. 18B). Rather, a moderate increase (74%) of
SOD1 protein was seen in ML-1 cells at 10 h. Thus, the increase of
SOD mRNA expression likely reflected a cellular response to
O.sub.2.sup.- accumulation due to SOD inhibition by 2-ME. To
confirm the accumulation of O.sub.2.sup.- in 2-ME-treated cells, we
used hydroethidine, a compound specifically converted by
O.sub.2.sup.- to highly fluorescent ethidium, to measure the
cellular O.sub.2.sup.- contents by flow cytometry analysis. FIG.
17C shows that treatment of ML-1 cells with 1 .mu.M 2-ME for 5 h
caused an increase of O.sub.2.sup.- from 4.3 to 6.3 arbitrary
units. Incubation of CLL cells with 30 .mu.M 2-ME also led to a
substantial O.sub.2.sup.- increase (226.+-.37%, FIG. 18D).
[0214] An in vitro assay for SOD activity was used to test the
direct effect of 2-ME on purified CuZnSOD and MnSOD. The
xanthine/xanthine oxidase system was used to generate
O.sub.2.sup.-, which reacted with XTT to produce a reddish product
(470 nm). Addition of CuZnSOD to the reaction caused a dismutation
of O.sub.2.sup.- and reduced OD.sub.470 nm in a
concentration-dependent manner (FIG. 19A). 2-ME prevented the
reduction of absorbance at 470 nm in the presence of SOD (FIG.
19B), indicating that 2-ME inhibited SOD activity. The inhibition
of human CuZnSOD, bovine CuZnSOD, and the E. coli MnSOD by 2-ME was
concentration-dependent (IC.sub.50.congruent.20 .mu.M, FIG. 19B).
Furthermore, 2-ME (1-100 .mu.M) showed little effect on xanthine
oxidase (FIG. 19C), DNA polymerase .alpha., or alkaline phosphatase
(FIG. 19D), suggesting that inhibition of SOD by 2-ME is likely
specific.
[0215] Four structurally related estrogen derivatives were compared
with 2-ME to investigate the chemical basis for inhibition of SOD.
As shown in FIG. 20, 2-ME (#1), 2-hydroxyestradiol (#4), and
2-methoxyestrone (#5), which have an --OH or --OCH.sub.3 group at
the 2-carbon, substantially inhibited SOD activity and induced DNA
fragmentation. In contrast, 17.beta.-estradiol (#2) and estrone
(#3) lacking the 2-carbon modification showed minimal activity
against SOD and caused little DNA fragmentation. It appeared that a
2--OH or 2-OCH.sub.3 modification was important for inhibiting SOD
and inducing apoptosis.
[0216] If inhibition of SOD were critical for the cytotoxic action
of 2-ME, one would expect that a change in SOD expression would
alter cellular sensitivity to 2-ME. When human ovarian cancer cells
(A2008) was infected with an adenoviral vector containing
full-length CuZnSOD sequence (Ad.CuZnSOD), substantial increases of
SOD1 protein were detected at 24 h and 48 h (FIG. 21A, lanes 2 and
3). This overexpression led to a decrease of 2-ME-induced
apoptosis, as evidenced by a reduction of nucleosomal DNA
fragmentation (FIG. 21A). In contrast, infection with the control
viral vector did not affect SOD expression (lane 4) or drug
sensitivity. MTT assay also revealed a partial protection of the
2-ME-treated cells by transduction with Ad.CuZnSOD or Ad.MnSOD
(FIG. 21B). Colony formation assay in another cell line (H1299)
further confirmed this protective effect (FIG. 21C).
[0217] Synthetic antisense S-oligos against SOD1 and SOD2 were used
to suppress SOD expression, and their effect on cellular
sensitivity to 2-ME was then evaluated. As shown in FIG. 21D,
incubation of A2008 cells with 10 .mu.M each of the anti-SOD
S-oligos for 48 h led to a moderate decrease of SOD1 and SOD2
protein. This was associated with an increase of 2-ME-induced
cytotoxicity. A longer incubation (72 h) with 20 .mu.M anti-SOD
S-oligos resulted in a greater enhancement of 2-ME activity.
Treatment with scrambled S-oligos did not alter the SOD protein
levels nor did the cellular sensitivity to 2-ME change (FIG. 21D).
Significant enhancement of the activity of 2-ME (0.3 .mu.M,
p=0.0059; 1 .mu.M, p=0.0452) by anti-SOD1 S-oligo was also observed
in H1299 cells (FIG. 21E). The role of SOD inhibition by 2-ME in
causing apoptosis was further evidenced by experiments with
antioxidants. When HL-60 cells were incubated with 1 .mu.M 2-ME in
the presence of the O.sub.2.sup.- scavenger ambroxol or antioxidant
N-acetylcysteine, the 2-ME-induced apoptosis was significantly
reduced (FIG. 21F). The protective effect of N-acetylcysteine was
also observed in CLL cells using the MTT assay.
[0218] Because mitochondria are a major source of superoxide
(O.sub.2.sup.-) production, we reasoned that inhibition of SOD by
2-ME might cause mitochondrial damage due to free radical attack on
the membrane phospholipids. To test this possibility, the integrity
of mitochondrial membranes of 2-ME-treated cells was examined by
measuring their ability to retain rhodamine-123, an fluorescent dye
used to indicate the loss of mitochondrial transmembrane potential.
Under the experimental conditions (rhodamine-123, 5 .mu.g/ml, 30
min), the control HL-60 cells retained 70-100 arbitrary units of
fluorescence (FIG. 23). During the first 6 h of 2-ME incubation, no
obvious loss of dye retention was observed. However, significant
loss of membrane integrity was seen after prolonged exposure to
2-ME (14 h), as evidenced by a shift of the fluorescence intensity
to much lower levels (20-40 units). By 22 h, almost all the cells
had lost their ability to retain rhodamine-123 in the
mitochondria.
[0219] It is known that cytochrome c is normally located in
mitochondria and its release to cytosol triggers apoptosis. Thus,
the possibility that the mitochondrial membrane damage by 2-ME
might result in the release of cytochrome c was tested. As shown in
FIG. 24, no cytochrome c was present in cytosol during the first 10
h. However, significant amounts of cytosolic cytochrome c were
detected at 12 and 14 h. This was in agreement with the time course
of mitochondrial membrane damage. In separate experiments, it was
observed that 2-ME-induced apoptosis occurred in the presence of
cycloheximide (5-50 .mu.g/ml) or actinomycin D (1 .mu.g/ml),
suggesting that induction of apoptosis by 2-ME did not require the
synthesis of new RNA or protein. This is consistent with the
mechanism of action of 2-ME in causing a free radical-mediated
damage to mitochondria membrane and release of cytochrome c, which
then activated the apoptotic cascade.
[0220] In the leukemia cell lines tested, 2-ME induced apoptosis at
concentrations of 1-10 .mu.M, whereas the IC.sub.50 for SOD
inhibition in vitro was approximately 20 .mu.M. This apparent
discrepancy was likely due to a concentrating uptake of 2-ME by the
cells. To confirm this, we incubated leukemia cell lines, primary
CLL cells, and normal lymphocytes with 1 .mu.M [.sup.3H]2-ME, and
determined the intracellular 2-ME concentrations. As shown in FIG.
22A, the drug was concentrated by approximately 10-fold in most
cases during the 5-h incubation. HPLC analysis of the extracts from
ML-1, HL-60 and CLL cells incubated with [3H]2-ME revealed no
significant drug metabolites (FIG. 22B). 2-ME appeared as the only
major peak (14.1 min) in all samples; there were two small peaks
barely detectable at 11.8 and 17.6 min.
Example 2
[0221] Based on our discovery that Superoxide dismutase (SOD) is a
key target of 2-methoxyestradiol (2-ME) in causing apoptosis of
cancer cells, we have further designed the following combination
strategies to enhance anticancer activity.
[0222] The first strategy involved a pharmacological approach to
increase the generation of intracellular O.sub.2.sup.- by rotenone
in combination with 2-ME to further block the elimination of
O.sub.2.sup.-, and thus enhance the free radical-mediated damage to
the cells. This strategy is shown in FIG. 23. Rotenone, an
inhibitor of mitochondrial enzyme complex I, inhibits the transport
of electron and cause a leak of electron from complex I to form
O.sub.2.sup.-. We have established a flow cytometry-based method to
quantitate cellular O.sub.2.sup.- contents. As shown in FIG. 24,
incubation of HL-60 cells with 0.25 .mu.M of 2-ME or rotenone led
to an increase of cellular O.sub.2.sup.- Combination of both
compounds caused a further O.sub.2.sup.- accumulation in the cells.
When sub-toxic concentrations of 2-ME and rotenone were combined,
substantial cytotoxic activity against HL-60 leukemia cells was
observed. This synergistic effect was demonstrated by nucleosomal
DNA fragmentation assay (FIG. 25), by flow cytometry analysis (FIG.
26), and by immunoblotting of PARP cleavage (FIG. 27). The killing
of leukemia cells by the combination of 2-ME and rotenone was
independent of cell cycle, as evidenced by depletion of cells from
all phases of the cell cycle and the appearance of sub-G1
population (FIG. 26). This is consistent with the free
radical-mediated damage to the mitochondrial membranes as a key
mechanism of apoptosis induction. We further demonstrated that this
synergistic effect was not due to change in cellular nucleotide
pools (FIG. 28).
[0223] Importantly, we discovered that rotenone, by diverting the
electron from complex I in the mitochondria to oxygen to form
O.sub.2.sup.-, also changed the redox status of cytochrome c, which
remained in oxidized form (Fe.sup.3+). The oxidized form of
cytochrome c was shown to be a more effective activator of
apoptotic cascade, as demonstrated by the cleavage of caspase-3 in
a cell free system (FIG. 29). Incubation of cytochrome c with
isolated nuclei also showed that the oxidized form of cytochrome c
was more effective in causing nucleosomal DNA fragmentation (FIG.
30). The enhancement of 2-ME activity by combination with rotenone
was also observed in H1299 cells, using a separate end point
(colony formation, FIG. 31).
[0224] The important role of free radical in mediating the
cytotoxic effect of 2-ME was further demonstrated by the use of an
antioxidant N-acetylcysteine (NAC). As shown in FIG. 32, incubation
of primary leukemia cells from a CLL patient with 2-ME in vitro
caused a significant accumulation of cellular O.sub.2.sup.-, and a
loss of cell survival. Addition of NAC reduced the accumulation of
cellular O.sub.2.sup.-, and protected the cells from the toxic
effect of 2-ME. These data support the conclusions that 2-ME causes
cell death by a free radical-mediated mechanism as a consequence of
SOD inhibition.
[0225] The second combination strategy to enhance anticancer
activity involved combination of 2-ME with gamma rays (ionizing
radiation, IR). The rationale for such a combination is shown in
FIG. 33. It is known that ionizing radiation causes a generation of
free radicals in the cells. Addition of 2-ME will cause an
inhibition of SOD activity and thus compromise the cell's ability
to cope with the free radicals generated by IR. This is expected to
cause a greater killing of cancer cells. Indeed, the data from
colony formation experiments in human lung cancer cells (H1299)
support this hypothesis. FIG. 34 demonstrates the synergistic
activity of 2-ME (1 .mu.M, 24 h) and IR (various doses). The number
inside each column indicates the observed surviving colonies. The
number above each column shows the expected % survival, assuming
that 2-ME and gamma radiation had an additive effect. In fact, the
observed cell killing was greater than the expected additive effect
under each combination condition.
Example 3
[0226] 2-Methoxylestradiol (2-ME) was combined with other agents
for cancer therapeutics. These data support the original
mechanism-based combination strategies and provide specific
combinational therapies that are effective in cancer treatment. The
data described herein were obtained in experiments with primary
leukemia cells isolated from blood samples obtained from patients
with chronic lymphocytic leukemia (CLL).
[0227] Combination with Sodium Arsenate.
[0228] Arsenic trioxide is known to possess anticancer activity and
has been approved for use in clinical treatment of certain type of
leukemia. Induction of free radical generation in the cells is
thought to contribute to its cytotoxic activity against cancer
cells. It is hypothesized that combination of arsenate (to enhance
cellular free radicals) and 2-ME (to inhibit the elimination of
superoxide radicals) would increase their anticancer activity. As
shown in FIGS. 39-40, the combination of arsenate and 2-ME
substantially increased the cytotoxic activity against primary
human leukemia cells from CLL patients in vitro. It should be noted
that the CLL cells from these two patients were relatively
sensitive to incubation with either 2-ME alone or arsenate alone.
The combined effect appears to be additive.
[0229] Importantly, leukemia cells isolated from difference
patients show different sensitivity to 2-ME. The heterogeneous
response to drug treatment in primary cancer cells from different
patients requires new strategies to overcome drug resistance.
Inventors tested if the combination of 2-ME with arsenate may
overcome cell's resistance to 2-ME. Primary leukemia samples from
two different CLL patients were identified to be resistant to 2-ME
(up to 30 .mu.M) in vitro. As shown in FIGS. 41-42, combination of
arsenate with 2-ME (15 .mu.M, FIG. 41; 30 .mu.M, FIG. 42) caused
significant loss of viability in the CLL cells, which were
insensitive to either drug alone. Thus, such drug combination may
have a potential to achieve therapeutic activity for patients who
are insensitive to treatment with 2-ME alone.
[0230] Combination with Rituxan
[0231] Rituxan (or rituximab) is an antibody specific for CD20
antigen on B lymphocyte, and is used in the clinical treatment of
certain B cell lymphoma. This agent was test for its combination
effect with 2-ME in primary leukemia B cells (CLL) in vitro.
Incubation of B CLL cells with various concentrations of Rituxan
alone caused an increased expression of c-myc protein, but did not
result in any significant killing of the CLL cells. There was only
a moderate activity against CLL cells when Rituxan was combined
with 2-ME at a fix ratio (2-ME/Rituxan=0.06). The results showed
that Rituxan did not significantly enhance the activity of 2-ME
either in cells sensitive to 2-ME alone or in cells resistant to
2-ME.
[0232] Combination with All-Trans Retinoic Acid (ATRA)
[0233] All-trans retinoic acid is an anticancer agent used in
clinical treatment of cancer, especially hematological
malignancies. Since retinoid derivatives have been shown to cause
an increase of cellular free radicals, we tested if combination of
2-ME with ATRA may result in an enhanced anti-leukemia activity. As
shown in FIG. 46, incubation of CLL cells with 2-ME and ATRA
resulted in a significant increase of cellular superoxide content,
and caused a synergistic activity against CLL cells. The dashed
lines above the bars indicate the predicted additive effect. The
observed anti-leukemia activity was clearly more than the additive
effect of both compounds.
[0234] Conclusions
[0235] Combinations of 2-ME with arsenic compounds or retinoic acid
derivatives will enhance anticancer activity and overcome drug
resistance to 2-ME. Such combination can have clinical implications
for cancer treatment.
[0236] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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Sequence CWU 1
1
6 1 27 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Primer 1 acgcacacgg ccttcgtcgc cataact 27 2 25 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Primer 2 gcacactgcc cggctcaaca tgctg 25 3 23 DNA Artificial
Sequence Description of Artificial Sequence Synthetic Primer 3
acgaaggccg tgtgcgtgct gaa 23 4 23 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Primer 4 accacaagcc
aaacgacttc cag 23 5 24 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Primer 5 ccatcaatga ccccttcatt gacc
24 6 23 DNA Artificial Sequence Description of Artificial Sequence
Synthetic Primer 6 gaaggccatg ccagtgagct tcc 23
* * * * *